The heart endothelium is the most significant blood clearing tissue in species of the bony fish family Poeciliidae and Gadidae, whereas the kidney plays important roles in the uptake of scavengers from the blood stream in salmonid species. Clearance of the blood in characid species is still poorly understood. Therefore, the aim of the present study was the analysis of the endocytotic capability and capacity of various tissues including cardiac endothelium in black tetra, Gymnocorymbus ternetzi (Boulenger) to take up foreign ferritin from the blood stream to establish the most significant blood-clearing organs and tissues in this species. Previous studies have shown that large amounts of intraperitoneally injected horse-spleen ferritin are taken up by cardiac endothelial cells in platyfish, Xiphophorus maculatus, family Poeciliidae. Electron microscopial studies have shown that horse ferritin is tightly packed in huge membrane-bound bodies, which often occupy the entire cellular volume in these tissues. Recently, it was shown that large amounts of Prussian blue precipitate in ferritin-filled bodies in cardiac endothelial cell layers in platyfish after treatment with acid ferrocyanide. Intracellular Prussian blue material appeared as deep-blue granular precipitates in these tissues and when such precipitations were present extracellularly they appeared to be amorphous, irregularly shaped and very faintly stained. In the present study, we have applied the same Prussian blue technique to visualize intracellular bodies of horse-spleen ferritin, i.e., we have used this technique to study the uptake of foreign ferritin in tissues of a number of organs in black tetras.

Twenty specimens of black tetra, G. ternetzi (Boulenger, 1895), 1–2 years old, approx. 0.7 g in weight and 3 cm in length, were kept in a well-aerated aquarium at 22°C, regularly fed with TetraMin (Tetra Werke, Melle, Germany) and were injected intraperitoneally with 0.02–0.04 ml of a 10% solution of horse-spleen ferritin (Sigma, St. Louis, MO, USA) by means of 0.5 ml tuberculin syringes (Becton Dickinson, New Jersey, NJ, USA). After 0.5, 1, 2, 3, 5, 6, 7, 8, 9 and 24 h, fish were killed with an overdose of chlorobutanol, and head kidney, trunk kidney, heart, gills, spleen, pancreas and liver were quickly excised and fixed at 4°C in 4% formaldehyde, made from paraformaldehyde within 24 h before use, in phosphate buffer, pH 7.4. The same tissues from eight untreated black tetras were also fixed for control studies or histology. After washing in buffer, the tissues were dehydrated through an ethanol series, treated with xylene, embedded in paraffin wax and sectioned (thickness, 4 ?m). Dewaxed sections were incubated with a ferrocyanide solution, made by dissolving 2 g potassium ferrocyanide in 100 ml 0.75 M hydrochloric acid solution, in order to visualize ferritin iron ions. Then, sections were treated either with 1% aquatic solutions of neutral red and eosin or with Mallory dye. Sections from untreated specimens were stained as described above or with Heidenhain’s Azan or Mayer’s haemalum/eosin. As the spleen of black tetra is always tightly packed with red blood cells, preparation of high-quality sections is difficult. However, we were able to obtain good-quality sections when the entire spleen had been fixed in a solution composed of 100% ethanol, 37% formaldehyde and 100% acetic acid in a volume ratio 85:10:5, instead of buffered formaldehyde solution. These sections were used to control and confirm findings in splenic tissue after fixation with buffered formaldehyde.

The head kidney in black tetra appeared to be rich in neutrophilic granulocytes and red blood cells. The trunk kidney consisted mainly of tightly packed renal tubules. The spleen was tightly packed with matured bloods cells, whereas ellipsoids as well as melano-macrophages were scarce; melano-macrophage centres were not observed at all. In the head kidney of ferritin-injected specimens, numerous cells with diameters up to 20 ?m were tightly packed with yellow-brown granules, with a diameter of 1–2 ?m, when the period of time between injection and sacrifice was over 2 h. When this tissue was treated with acid ferrocyanide, the cellular granules were entirely filled with Prussian blue precipitates Smaller cells, with a diameter of approx. 10 ?m, and filled with yellow-brown granules or Prussian blue precipitates, were also observed in trunk kidney, gills and peripheral connective tissue of pancreas and spleen at 2 h or more after injection, but these cells were always scarcely present. These granules were not present in neutrophilic granulocytes in kidney, eosinophilic granulocytes in gills or in the endothelia of heart, liver, kidney and spleen. Amorphous, irregularly shaped and faintly stained Prussian blue material was regularly present in the cavity of hearts after treatment with acid ferrocyanide.

Figure 1. (a) The head kidney in black tetra, G. ternetzi, is rich in neutrophilic granulocytes (arrow) and red blood cells. (b–d) Head kidney from black tetras after an intraperitoneal injection of horse-spleen ferritin at 8 h before sacrifice; endocytotic cells are packed with bodies of yellow-brown ferritin in (c), whereas tissues in (b) and (d) have been treated with acid ferrocyanide and large amounts of Prussian blue are precipitated in the ferritin-containing bodies in endocytotic cells. Neutrophilic granulocytes (arrows) do not contain ferritin and Prussian blue material. (e, f) Trunk kidney and gill, respectively, of black tetra after an intraperitoneal injection with horse-spleen ferritin at 8 h before sacrifice; the tissues have been treated with acid ferrocyanide, and contain some cells filled with Prussian blue precipitations. The eosinophilic granulocyte (arrow) within the gill does not contain such precipitations. (g) The spleen of black tetra, sacrificed at 8 h after ferritin injection, is tightly packed with matured blood cells. The tissue has been treated with acid ferrocyanide; Prussian blue precipitations (arrowhead) are small and few in number. (h) The heart atrium of black tetra, sacrificed at 8 h after ferritin injection. The tissue has been treated with acid ferrocyanide, but lacks Prussian blue granules. Amorphous and very faintly stained Prussian blue material is present in the heart cavity. (a–c, e, g) Mallory staining, (d) neutral red staining, (f, h) neutral red/eosin staining. Scale bar, 20 ?m.

Yellow-brown granules or Prussian blue precipitations were totally absent in control tissues.

In general, macrophages and sinusoidal endothelial cells are cell types in head kidney, trunk kidney and spleen that participate in clearance of scavenger and foreign macromolecules from the circulation of bony fish. However, in plaice (Pleuronectes platessa), Atlantic cod (Gadus morhua) and platyfish (Xiphophorous maculatus) heart endothelium appears to be the most important blood clearing tissue. The present study reveals that large amounts of horse-spleen ferritin are taken up by a high number of large cells in the black tetra head kidney, whereas these endocytotic cells are scarce and smaller in trunk kidney, spleen and gills, and ferritin-uptake was not observed at all in heart and liver. The present findings are rather different from those recently reported for platyfish, in which trunk kidney and spleen were able to take up horse-spleen ferritin, and large amounts of ferritin were taken up as well by cardiac endothelial cell layers in this species, particulary in the atrium. We suggest that practically all blood clearance in black tetras occurs in the head kidney.

The present study demonstrates that the spleen in black tetras consists mainly of tightly packed matured blood cells, whereas in platyfish and some other teleosts studied until now, spleen contains numerous ellipsoids and large areas of white blood cells, including well-developed melano-macrophage centres. However, in rainbow trout (Salmo gairdneri), carp (Cyprinus carpio) and the Antartic teleost Chionodraco hamatus the spleen consists mainly of red blood cells as well, i.e., the spleen in these species displays a similar structure as in black tetra. Thus, the spleen in teleosts seems to have a more variable structure and functional roles than anticipated until now. Therefore, we suggest that the spleen may function mainly as reservoir for red blood cells in the black tetra.